During organogenesis the internal organs of the embryo display significant left-right asymmetry (L-R). A sequence of events for the establishment of this asymmetry includes: a) the initial breaking of symmetry, which leads to the establishment of specific patterns of gene expression in and around the embryonic organizer; b) the relay of L-R positional information from the organizer to the lateral plate mesoderm, and c) the transfer of L-R information to the organ primordia that leads to the elaboration of specific programs of asymmetric morphogenesis. A variety of genes involved in these three processes have been identified in recent years. Our preliminary results indicate that, additionally, the Notch pathway, a major developmental signaling pathway, may have an important role in L-R determination. The main focus of this proposal will be to study the role of the Notch signaling pathway during each one of the three events described above, and its interaction with other known genes implicated in L-R determination. We plan to tackle these questions using modem visualizing techniques in vivo, in parallel with genetic interventions in model animals such as chick, zebrafish and mouse. Specifically, we propose to: 1) study the role of cilia in breaking L-R symmetry and their regulation by the RFX family of transcription factors in zebrafish, chick and mouse embryos; 2) Perform gain- and loss-of-function experiments of various components of the Notch pathway (chick, mouse and zebrafish) and establish its relationship with nodal cilia movement and flow in mice and Ca 2+ in zebrafish; 3) analyze the spatio-temporal expression of genes known to be involved in L-R asymmetry in zebrafish wild-type and mutant embryos, in an effort to obtain a mathematical model of the establishment of LR asymmetry in the zebrafish embryo; and 4) identify novel genes upstream of Nodal, Notch and Pitx2 by means nf large-scale mutagenesis screens of transgenically engineered zebrafish embryos. Until now, very few genes have been implicated in the etiology of human laterality disorders. The study of the early stages of L-R determination and the identification of new genes involved in various vertebrate model organisms would undoubtedly shed new light on the genetic factors contributing to laterality defects in humans.